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Results for “Intracellular quantification”

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PubMed1997

Thrombomodulin, a functional surface protein on human keratinocytes, is regulated by retinoic acid.

Thrombomodulin, a major anticoagulant proteoglycan of the endothelial cell membrane, is a thrombin receptor that acts as a cofactor for protein C activation. It has previously been shown that thrombomodulin, present in human epidermis and in lysates of cultured keratinocytes, is implicated in cellular differentiation during mouse fetal development. The role of retinoic acid in keratinocyte differentiation prompted us to study retinoic acid regulation of thrombomodulin expression in primary cultures of keratinocytes isolated from adult human skin, grown at low (undifferentiated keratinocytes) and normal calcium levels (differentiated keratinocytes). Thrombomodulin antigen levels and total and surface activities were measured in cultures without and with retinoic acid. Thrombomodulin mRNA visualized by in situ hybridization was quantified by computer-based image analysis. Functional thrombomodulin was expressed on the surface and in the cytoplasm of cultured human keratinocytes regardless of the calcium concentration. In contrast, retinoic acid induced significant increases in the total antigen level and in surface and intracellular thrombomodulin activities only in keratinocytes grown in a low-calcium medium. In these undifferentiated keratinocytes, quantification of mRNA transcripts showed a threefold increase after retinoic acid stimulation. Thus, functional thrombomodulin is a human keratinocyte surface protein whose expression is controlled through the keratinocyte differentiation program and is modulated in vitro by retinoic acid.

Adult↗

PubMed2003

Nonradiometric HPLC measurement of 13(S)-hydroxyoctadecadienoic acid from rat tissues.

A major bioactive metabolite of linoleic acid formed by the action of 15-lipoxygenase-1 is 13(S)-hydroxy-cis-9, trans-11-octadecadienoic acid (13(S)-HODE). 13(S)-HODE is an important intracellular signal agent and is involved in cell proliferation and differentiation in various biological systems. Separation and quantification of 13(S)-HODE from biological materials has previously been achieved only by using radiolabeled linoleic acid as the substrate and two serially connected or two separate HPLC columns to achieve separation of 13(S)-HODE. In the current method, separation and quantification of 13(S)-HODE was achieved by use of a normal-phase HPLC and a solvent system containing hexane/isopropanol/acetonitrile/acetic acid (800/8/30/1, v/v) using isocratic elution with detection at 235 nm. With the currently described method, good separation from unreacted interfering compounds and quantification for 13(S)-HODE were achieved within 35 min with a minimum detection limit of 0.5 ng per injection.

2-Propanol↗

PubMed1987

Heart Na,K-ATPase activity in cardiomyopathic hamsters as estimated from K-dependent 3-O-MFPase activity in crude homogenates.

The hamster hereditary cardiomyopathy provides a unique model for the study of membrane abnormalities during chronic congestive heart failure. It is associated with intracellular calcium accumulation, mitochondrial calcification and cell necrosis. Previous studies have shown a decrease in Na,K-ATPase activity purified from ventricle sarcolemma. The present study demonstrates a decrease in K-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) activity from 1.93 to 1.30 mumol/g wet wt. or 33% in crude homogenates from the left ventricle of 7-months-old cardiomyopathic hamsters as compared to control animals. This represents an equivalent decrease in Na, K-ATPase activity. The values are several times higher than previously published for membrane fractions of myocardium from the hamster. Concomitantly, there was an increase in intracellular Na-concentration of the myocardium of 42% whereas the K-concentration was unchanged. The decrease in Na,K-pump concentration may be of importance for the increase in intracellular sodium and ensuing calcifying necrosis observed in the myocardium of cardiomyopathic hamsters. It is emphasized that quantification of the Na,K-ATPase or Na,K-pump should preferably be performed using crude homogenates.

Animals↗

PubMed2002

Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms.

Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus (P) removal. Considerable effort has been devoted to understanding the biochemical nature of enhanced biological phosphorus removal (EBPR) and it has been shown that intracellular polymer storage plays an important role in PAO's metabolism. The storage capacity of PAOs gives them a competitive advantage over other microorganisms present that are not able to accumulate internal reserves. Intracellular polymers stored by PAOs include polyphosphate (poly-P), polyhydroxyalkanoates (PHAs) and glycogen. Staining procedures for qualitative visualization of polymers by optical microscopy and combinations of these procedures with molecular tools for in situ identification are described here. The strengths and weaknesses of widely used polymer quantification methods that require destruction of samples, are also discussed. Finally, the potential of in vivo nuclear magnetic resonance (NMR) spectroscopy for on-line measurement of intracellular reserves is reported.

Bacteria, Aerobic↗

PubMed1995

Fluorometric measurement of the intracellular free Ca(2+)-concentration in the ciliate Didinium nasutum using Fura-2.

We developed an experimental approach to measure somatic and ciliary Ca(2+)-signals in the ciliate Didinium under voltage clamp conditions using the dye Fura-2. Intracellular pressure injection of Fura-2 molecules did not alter electrophysiological membrane properties besides an expected buffering effect. The intracellular free Ca(2+)-concentration was determined at 2 x 10(-7) M. During membrane excitation, this resting value increased in the cilia; a quantification was not feasible. Within the cell soma, however, the Ca(2+)-level was unchanged within the physiological range of the membrane potential (-70 mV to 0 mV). Increasing the driving force for Ca(2+)-ions via strong hyperpolarization (potentials negative to -200 mV) a centripetal increase in the somatic Ca2+ concentration was found. Our results support the hypothesis that Ca2+ is the intracellular messenger in rapid electromotor coupling in ciliates.

Animals↗

PubMed2002

Transfer of lipophilic markers from PLGA and polystyrene nanoparticles to caco-2 monolayers mimics particle uptake.

PURPOSE: The objective of this study was to evaluate nanoparticle uptake by the Caco-2 monolayer model in vitro. Special emphasis was placed on the localization and the quantification of the uptake of fluorescently labeled polystyrene and poly(lactic-co-glycolic acid) (PLGA) nanoparticles. METHODS: Intracellular fluorescence was localized by fluorescence and confocal laser scanning microscopy. Particle uptake was quantified either directly, by counting internalized nanoparticles after separation from the Caco-2 monolayers, or indirectly, by extraction of the lipophilic fluorescence marker. In vitro release studies of lipophilic markers from nanoparticles were performed in standard buffer systems and buffer systems supplemented with liposomes. RESULTS: Instead of uptake of polystyrene and PLGA nanoparticles by Caco-2 monolayers an efficient transfer of lipophilic fluorescence markers from nanoparticles into Caco-2 cells with subsequent staining of intracellular lipophilic compartments was observed. Whereas in standard buffer no release of fluorescent marker from polystyrene and PLGA nanoparticles was observed, the release studies using liposome dispersions as receiver revealed an efficient transfer of fluorescent marker into the liposome dispersion. CONCLUSIONS: The results suggest that the deceptive particle uptake is caused by a collision-induced process facilitating the transfer of lipophilic fluorescent marker by formation of a complex between the nanoparticles and the biomembranes. Diffusion of the marker within this complex into lipophilic compartments of the cell strongly affects quantitative evaluation of particle uptake.

Biological Transport↗

PubMed1988

Immunolocalization of matricial components during the early stages of chick embryonic liver development.

In the chick embryo, the first liver primordium is observed at the end of the second day of incubation. At 3 and 4 days, ultrastructural analysis of the primitive vascular spaces showed that the endothelial limiting plate was constituted by one or several cell layers. At the vascular pole of the hepatoblasts, mesenchymal cells and connective matrix, present as fibrillar and non fibrillar components, were closely associated. At 5 days, some vascular spaces were limited by a simple endothelial layer. The limiting plate was fenestrated and the connective matrix was reduced to rare collagen fibrils and fibers. Collagen types I, III, IV, procollagen type III, fibronectin and laminin were visualized in the perivascular spaces using immunoperoxidase labeling methods. These components were also detected in the endoplasmic reticulum of hepatoblastic, endothelial and mesenchymal cells. All these appeared to be involved in connective matrix synthesis. Comparing 4 and 5 days, we demonstrated that the number of cells showing intracellular labelling of matricial components dropped dramatically at 5 days, indicating a possible decrease of connective matrix synthesis. Quantification of parenchymal and vascular surfaces was carried out using a semi-automatic image analyzer on consecutive parasagittal sections chosen in the axial part of the embryonic liver. These measurements were performed in order to quantitate the vascular distribution pattern during early development of the liver. These combined immunomorphological studies and morphometrical analyses suggest that during embryogenesis of the liver the synthesis of connective matrix precedes and possibly initiates the vascular differentiation.

Animals↗

PubMed2000

Detection and quantification of free cytosolic inorganic phosphate and other phosphorus metabolites in the beating mouse heart muscle in situ.

The aim of this study was the quantification of inorganic phosphate (Pi) and other phosphorus metabolites by (31)P NMR spectroscopy in the mouse heart muscle in situ, beating at around 600 min(-1). Male adult Quacker-bush mice (mean weight 32 +/- 7 g) were anaesthetized, ventilated and placed in a temperature-controlled animal holder. A purpose-built (31)P NMR surface coil was positioned against the exposed left ventricular myocardium. Partial signal overlap of Pi with 2,3-DPG from chamber blood was minimized using a DEPTH pulse sequence (180 degrees -90 degrees -180 degrees -180 degrees -acq.). Quantification of phosphorus metabolites was performed using an external standard positioned directly above the surface coil. We report for the mouse myocardium in situ an intracellular free [Pi] of <0.4 mM, pH of 7.32 +/- 0.1, free [Mg2+] of 0.41 +/- 0.1 mM, free [ADP] of 13 +/- 1.5 microM, [ATP] of 5 +/- 0.5 mM and [PCr] of 14 +/- 1.5 mM. The phosphorylation ratio (ATP/ADP Pi) was 1005 +/- 200 mM (-1) for a PCr/ATP ratio of 2.7 +/- 0.3. It was concluded that the detection of free [Pi] in the mouse myocardium in situ can be greatly enhanced using a DEPTH pulse sequence. Quantification of compounds using an external standard positioned directly above the surface coil gave comparable results to estimations using internal ATP that was quantified enzymatically. The close agreement between the external and internal methods indicates that ATP is 100% NMR visible in the mouse heart in situ.

Animals↗

PubMed2001

Quantification of annexin I in subcellular fractions of human neutrophils reveals an exclusive cytosolic localisation.

Annexin I is an abundant cytosolic protein in human neutrophils. Besides its intracellular location, annexin I is found as an extracellular protein and the pathway for secretion has been of interest since the protein lacks a signal sequence for secretion. It was recently shown that annexin I is stored in the secretory gelatinase granules of human neutrophils, suggesting that the protein might be released through a granule mobilisation and fusion process resembling classical secretion. In this study we have determined the intracellular localisation of annexin I in human neutrophils using subcellular fractionation, protein separation by SDS-PAGE and immunoblotting, and show that virtually all annexin I is localised in the cell cytosol.

Annexin A1↗

PubMed1993

Detection of intracellular interferon-gamma by light microscopy using an immunoperoxidase technique: correlation with the corresponding mRNA and protein product.

Identifying individual cytokine-producing cells may help to acquire insight into immunological processes. This study was designed to adapt a technique for the detection of individual cytokine-producing cells from an immunofluorescence to an immunoperoxidase staining procedure. The production of interferon-gamma (IFN-gamma) by anti-CD3-activated cloned human T cells was used as a model system. After the conditions for the staining procedure were optimized, the immunoperoxidase technique was slightly more sensitive than the immunofluorescence technique. The intracellular staining for IFN-gamma was preceded or paralleled by IFN-gamma mRNA production and followed by accumulation of IFN-gamma in the supernatant. It is concluded that intracellular IFN-gamma can easily be detected using an immunoperoxidase procedure. This procedure is highly sensitive and allows quantification of the production of multiple cytokines by counting the percentage of positively staining cells.

Cytokines↗

PubMed2001

Transglutaminase activity is involved in polyamine-induced programmed cell death.

Natural polyamines, i.e., putrescine, spermidine, and spermine, are ubiquitous molecules essential for cell proliferation and differentiation. In the present study, the effect of polyamines on primary cultures of bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and a human melanoma cell line was examined. While in the absence of fetal calf serum (FCS) polyamines had no effect on viability, in the presence of FCS spermidine and spermine, at concentrations close to physiologic levels, induced a dose-dependent cell death, whereas putrescine was ineffective. RASMCs were significantly more sensitive than other cells. FACS analysis, oligo-nucleosome ELISA, Hoechst nuclear staining, and Annexin V-FITC quantification showed that cell death was likely due to apoptosis. Cells exposed to spermidine showed a marked increase of intracellular transglutaminase (TGase) activity ( approximately 30-fold over control). Inhibitors of polyamine oxidation or inhibitors of TGase activity prevented polyamine-induced apoptosis. Moreover, tissue TGase overexpression significantly increased cell sensitivity to polyamine, suggesting that this effect is likely related to enhanced intracellular TGase activity. These data indicate that polyamines may modulate cell viability through a novel TGase-dependent process.

Animals↗

PubMed1977

Spin-lattice relaxation times for 13C in isotope-enriched glycine accumulated in frog muscle.

Spin-lattice relaxation times (T1's) of 13C-enriched glycine accumulated in frog muscles were determined at 1 degrees C by the inversion-recovery (180 degrees -tau-90 degree pulse sequence) method and compared with the values obtained in free solution. The value of T1 for the alpha-13C nucleus of glycine in the tissue was 50% of that obtained in free solution. The observed value for T1 in the tissue was not concentration-dependent, and no difference in chemical shift was observed between tissue and free solution. Quantification of the area under the glycine peak suggested that the observed signal represents at least 80% of the intracellular glycine. An average nuclear Overhauser enhancement of 2.83 for intracellular glycine indicates that the relaxation mechanism within the cell is predominantly dipolar, as in free solution. The value of T1 for the 13C' nucleus of glycine in the tissue was 67% of that in a solution of similar concentration. A quantitative analysis of the findings suggests that the observed difference in the value of T1 between tissue and free solution results from a difference in viscosity. The data provide no evidence either for special organization of intracellular water or for glycine binding. It is proposed that intracellular diffusion coefficients may be determined from measurements of 13C T1's of 13C-enriched intracellular solutes.

Animals↗

PubMed2000

Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms.

After hepatitis B virus (HBV) infection, liver injury and viral control have been thought to result from lysis of infected hepatocytes by virus-specific cytotoxic T cells. Patients are usually studied only after developing significant liver injury, and so the viral and immune events during the incubation phase of disease have not been defined. During a single-source outbreak of HBV infection, we identified patients before the onset of symptomatic hepatitis. The dynamics of HBV replication, liver injury, and HBV-specific CD8+ and CD4+ cell responses were investigated from incubation to recovery. Although a rise in alanine transaminase (ALT) levels was present at the time of the initial fall in HBV-DNA levels, maximal reduction in virus level occurred before significant liver injury. Direct ex vivo quantification of HBV-specific CD4+ and CD8+ cells, by using human leukocyte antigen (HLA) class I tetramers and intracellular cytokine staining, showed that adaptive immune mechanisms are present during the incubation phase, at least 4 weeks before symptoms. The results suggest that the pattern of reduction in HBV replication is not directly proportional to tissue injury during acute hepatitis B in humans. Furthermore, because virus-specific immune responses and significant reductions in viral replication are seen during the incubation phase, it is likely that the immune events central to viral control occur before symptomatic disease.

Acute Disease↗

PubMed2001

Nucleocytoplasmic transport in human astrocytes: decreased nuclear uptake of the HIV Rev shuttle protein.

Astrocytes are cellular targets for the human immunodeficiency virus (HIV) that limit virus production, owing, at least in part, to the diminished functionality of the viral post-transcriptional stimulatory factor Rev. To understand the trafficking process in astrocytes, we compared nucleocytoplasmic transport of Rev and various proteins with well-characterized nucleocytoplasmic transport features in human astrocytes and control cells (HeLa). Localization and trafficking characteristics of several cellular and viral proteins, as well as nuclear trafficking of classical peptide signals upon microinjection were similar in both cell types, indicating maintenance of general features of nucleocytoplasmic transport in astrocytes. Quantification of fluorescence in living cells expressing Rev fused to green fluorescent protein (GFP) indicated a strong shift in intracellular distribution of Rev in astrocytes, with 50-70% of Rev in the cytoplasm, whereas the cytoplasmic proportion of Rev in HeLa cells is around 10%. The dynamics of nucleocytoplasmic trafficking of Rev were compared in astrocytes and Rev-permissive cells by monitoring migration of Rev-GFP in cell fusions using highly sensitive time-lapse imaging. Nuclear uptake of Rev was dramatically retarded in homo-polykaryons of astrocytes compared with control cells. Diminished nuclear uptake of Rev was also observed in hetero-polykaryons of Rev-permissive cells and astrocytes. These results indicate that astrocytes contain a cytoplasmic activity that interferes with nuclear uptake of Rev. Our studies suggest a model in which Rev is prevented from functioning efficiently in astrocytes by specific alterations of its nucleocytoplasmic trafficking properties.

Amino Acid Sequence↗

PubMed1983

Immunoreactive prolactin in subcellular fractions from bovine mammary tissue.

Mammary tissue from lactating Holsteins at slaughter was homogenized and fractionated into subcellular compartments. Bovine prolactin was quantified in each subcellular fraction and in preslaughter milk by radioimmunoassay procedures validated for this purpose. Prolactin concentrations were ng prolactin/mg protein for all samples. Immunoreactive prolactin was in rough endoplasmic reticulum (24.8 ng/mg), Golgi apparatus (18.8 ng/mg), and secretory vesicle (10.2 ng/mg). All of these concentrations exceeded that in samples of milk (2.7 ng/mg). Nuclear fractions contained a material that reacted in the prolactin radioimmunoassay, but criteria for valid quantification could not be satisfied. These results confirm prolactin inside bovine mammary cells, and they are consistent with an intracellular pathway for transfer of prolactin from plasma into milk.

Animals↗

PubMed1999

[Cytokines in children with immunodeficiencies].

The underlying immunological defect in humoral immunodeficiency with decreased production of immunoglobulin and normal level of circulating B cells (IgA deficiency, transient hypogammaglobulinemia of infancy, common variable immunodeficiency) remains unknown. There is evidence that B cells maturation and differentiation is regulated by cytokines and hence aberrant cytokine production may be involved in the pathogenesis of these diseases. Among interleukins, IL-1, IL-4, IL-6 and IL-10 may each play a role in regulation of B cell growth. Interferons (IFNs) have been described as having both positive and negative effects on B cell growth. The precise role of tumor necrosis factors (TNF alpha and beta) remains obscure. In the present study the in vitro cytokine production by peripheral blood mononuclear cells (PBMC) from children with different forms of immunodeficiency, in particular with IgA deficiency and transient hypogammaglobulinemia of infancy, was evaluated. In the first stage of the study the release of IL-1, IL-6, IFN and TNF by PBMC was analysed in following groups of patients: transient hypogammaglobulinemia (n = 30), IgA deficiency (n = 29), Bruton's disease (n = 7), decreased proliferative response to mitogenes (n = 10), CD4+ lymphocytopenia (n = 8), CD8+ lymphocytopenia (n = 10). The concurrent control group consisted 52 sex- and age-matched children, in whom no immunological immunological abnormalities were detected. The release of bioactive IL-1, IL-6, IFNs and TNF was measured in the culture supernatants from PBMC stimulated with mitogens for 48 hours. While the release of bioactive IL-1, IL-6, and IFNs was comparable in all studied groups, the secretion of TNF was significantly increased in children with transient hypogammaglobulinemia and IgA deficiency. The next issue was determination of the type of TNF (alpha or beta) involved. The production of other cytokines important for the regulation of B cell function (IL-4, IL-10) was also assessed. Production of TNF alpha, TNF beta and IL-10 was significantly elevated in transient hypogammaglobulinemia. The data from the ELISPOT assay suggested, that in these patients also the number of cells secreting TNF alpha after PHA stimulation was increased. These results indicate, that elevated TNF alpha production was probably due to both an enhanced release and an increased number of circulating secreting cells. As the methods employed in the quantification of cytokine levels in culture supernatants do not allow identification of the cytokine producing cell, the studies on intracellular expression of cytokines were undertaken. The fluorochrome-labelled monoclonal antibodies against the cell surface markers and a given cytokine were used simultaneously to identify the cellular source of cytokine production. The intracellular IL-4 expression in CD4+ lymphocytes from patients with transient hypogammaglobulinemia was comparable to that of control while the number of CD4+ lymphocytes expressing TNF alpha, TNF beta and IFN gamma was elevated. The number of CD14+ cells (monocytes) producing of TNF alpha was comparable to the control. These results suggest that an excessive Th-1 type response may contribute to pathology of this disease. In patients with isolated IgA deficiency the significantly increased release of TNF alpha but not: IL-1, IL-4, IL-6, IL-10 and TNF beta was observed. The proportion of CD4+ lymphocytes that expressed TNF alpha was significantly increased while the number CD14+ cells staining for TNF alpha was unchanged. No changes in the expression of TNF type I and II receptors on PBMC were observed, which suggested that regulatory effects of TNF alpha and beta are associated rather with an increased production of these cytokines than an abnormal receptor expression. Some patients with transient hypogammaglobulinemia were followed-up and the serum level of IgG and production of TNF alpha, TNF beta, and IL-10 by their PBMCs was determined 6 to 12 months after first

Agammaglobulinemia↗

PubMed1991

Phosphorus-31 NMR spectra by the linear prediction z-transform method.

The linear prediction z-transform method (LPZAR) was applied to estimate 31P NMR spectra of perfused rat hearts. The spectra obtained by the LPZAR method showed sharper peaks with less noise for phosphate concentrations than those obtained by conventional fast Fourier transform. The LPZAR method provided a better estimation of intracellular phosphate concentrations in short acquisition time experiments in which condition Fourier transform spectra are not suitable for the quantification due to the poor resolution of FFT spectra. Utilizing the LPZAR method, rapid changes in phosphoric metabolites of the heart can be monitored during ischemia followed by reperfusion.

Animals↗

PubMed1981

Flow cytometry studies of intracellular adriamycin in multicell spheroids in vitro.

A marked gradient of Adriamycin uptake in cells of tissue-like multicell spheroids in vitro has been demonstrated by fluorescence photomicroscopy and flow microfluorimetry techniques. As expected, this gradient led to an increased net survival for cells from Adriamycin-treated spheroids relative to monolayers and markedly greater clonogenicity of central spheroid cells than external cells selected by fluorescence-activated cell sorting. Growth of cells as spheroids seemed to impart an additional degree of drug resistance relative to cells grown as monolayers, in that equal toxicity required greater intracellular fluorescence (and thus more Adriamycin) for the spheroid cells. The flow cytometry techniques thus provide a mechanism for quantification of Adriamycin penetration into spheroids and provide a method for selection of cells from various depths within the spheroid.

Animals↗